Method for purifying RNA
The present invention relates to methods for purifying RNA by chromatography under high salt conditions, e.g. by hydrophobic interaction chromatography.
1. A method for purifying RNA, comprising the steps of:
a) applying a sample containing in vitro transcribed RNA in an equilibration buffer having a high salt concentration to a support material that binds the RNA under said high salt concentration, wherein the support material comprises a binding ligand consisting of hydroxyl or sulfate groups and, wherein the equilibration buffer has said high salt concentration of at least about 150 mM;
b) optionally washing the support material with a washing buffer having a high salt concentration of at least about 150 mM; and
c) eluting the RNA from the support material with an elution solution.
2. The method according to claim 1 , wherein the equilibration buffer and/or the washing buffer has a salt concentration of 500 mM to 3 M.
3. The method according to claim 1 , wherein the equilibration buffer and/or the washing buffer comprises sodium chloride or ammonium sulfate.
4. The method according to claim 1 , wherein the equilibration buffer and/or the washing buffer comprises at least about 2 M NaCl.
5. The method according to claim 4 , wherein the equilibration buffer and/or the washing buffer comprises 20 mM HEPES-NaOH, pH 7.0, 2 M NaCl.
6. The method according to claim 1 , wherein the equilibration buffer and the washing buffer have the same composition and the same pH.
7. The method according to claim 1 , wherein the support material is a monolithic support material.
8. The method according to claim 1 , wherein the support material is a methacrylate polymer.
9. The method according to claim 1 , wherein the RNA is eluted by gradually decreasing the salt concentration of the elution solution.
10. The method according to claim 1 , wherein the elution solution does not contain a salt.
11. The method according to claim 1 , wherein the elution solution comprises 20 mM HEPES-NaOH, pH 7.0.
12. The method according to claim 1 , wherein the method comprises the steps of:
a) transcribing RNA from a template DNA in vitro;
b) applying a sample containing the in vitro transcribed RNA in an equilibration buffer having a high salt concentration of at least about 150 mM to a support material capable of binding the RNA under high salt conditions, wherein the support material comprises a binding ligand consisting of hydroxyl or sulfate groups;
c) washing the support material with a washing buffer having a high salt concentration of at least about 150 mM; and
d) eluting the RNA from the support material with an elution solution.
13. The method according to claim 12 , further comprising a step a1) of degrading the template DNA.
14. The method according to claim 13 , wherein the template DNA is degraded by treatment with DNase.
15. The method according to claim 12 , further comprising a step a2) of subjecting the in vitro transcribed RNA to a reverse phase-HPLC step.
16. The method according to claim 12 , wherein said RNA is suitable for preparing a pharmaceutical composition.
17. The method according to claim 2 , wherein the RNA is a mRNA.
18. A method for preparing a pharmaceutical composition comprising:
a) transcribing RNA from a template DNA in vitro;
b) applying a sample containing the in vitro transcribed RNA in an equilibration buffer having a high salt concentration of at least about 150 mM to a support material capable of binding the RNA under high salt conditions, wherein the support material comprises a binding ligand consisting of hydroxyl or sulfate groups;
c) washing the support material with a washing buffer having a high salt concentration of at least about 150 mM; and
d) eluting the washed RNA from the support material with an elution solution; and
e) formulating the eluted RNA into a pharmaceutical composition.
19. The method according to claim 18 , wherein the equilibration buffer and/or the washing buffer comprise about 500 mM to 3 M NaCl.
20. The method according to claim 17 , wherein the binding ligand contains hydroxyl groups.
21. The method according to claim 17 , wherein the binding ligand contains sulfate groups.